Project Summary/Abstract Model organisms, such as Saccharomyces cerevisiae, have proven effective in elucidating genes and metabolites that modulate lifespan and this knowledge forms a basis for understanding human aging and longevity. These studies have identified the Akt/PKB protein kinases as conserved regulators of aging. However, it is not entirely clear how they work. One focus of our research is the S. cerevisiae Sch9 protein kinase, an AKT homolog that regulates chronological life span (CLS). From in vitro studies we identified sphingolipid long chain bases (LCBs) and Pkh1, a homolog of human phosphoinositidedependent protein kinase 1 (PDK1), as the first upstream regulators of Sch9. In addition, we identified the first Sch9 substrates, Rli1, involved in translation initiation and ribosome biogenesis, and Ahp1, an alkyl hydroperoxide reductase that protects against oxidative stress. We will build upon these novel results and determine if phosphorylation of Rli1 and Ahp1by Sch9 plays a role in CLS. We will also determine in vivo if LCBs and Pkh1 act upstream to regulate Sch9 during chronological aging. Published data suggest that the TOR proteins also regulate Sch9 during aging and we will determine if this hypothesis is true. Although we have successfully identified Sch9 substrates, there are likely to be others and we propose to search for new substrates by classical biochemical strategies and newer automated but complementary strategies. In summary, the results of these studies will provide the much needed mechanistic understanding of how Akt (Sch9) activity is regulated in yeast and how it regulates CLS through downstream substrates. This knowledge will help to provide a molecular basis for understanding human aging and longevity.